Plant lipids have become indispensable in a number of industrial and nutritional applications. More importantly, plant lipids are used extensively for their nutritional value, which is determined by a plant's fatty acid composition. The value of any plant lipid is determined by its chemical structure, which is a result of a plant's metabolic processes. The chemical structure is characterized by varied degrees of unsaturation. Most vegetable oils from commercial plant varieties are composed primarily of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic acid (18:3) acids.
Numerous research efforts have shown lipids to play a major role in development of many diseases, especially cardiovascular disorders. Recent research has examined in great detail the role of saturated and unsaturated fatty acids in potentiating the risk of coronary heart disease. Previously, it was believed that mono-unsaturated fatty acids had no effect on serum cholesterol and coronary heart disease risk. On the other hand, saturated fatty acids were considered to contribute to coronary heart disease while poly-unsaturated fatty acids were supposed to lower the risk of the same. It is now known that intake of both mono-unsaturated and poly-unsaturated fatty acids is beneficial for the heart and overall health. Several recent human clinical studies suggest that diets high in mono-unsaturated and/or poly-unsaturated fat and low in saturated fat may reduce the “bad” (low-density lipoprotein or LDL) cholesterol while maintaining the “good” (high-density lipoprotein or HDL) cholesterol. For example, a study performed by Mensink et al. concluded that a diet rich in mono-unsaturated fatty acids was as effective as a diet rich in (n-6)poly-unsaturated fats in lowering “bad” cholesterol (Mensink et al., N Engl J Med 321(7)436-441, August 1989). Furthermore, another study found that a diet rich in poly-unsaturated fats has a similar effect on “good” cholesterol concentrations in the blood as a diet rich in mono-unsaturated fats (Dreon et al., JAMA 263(18):2462-2466, May 9, 1990). Animal studies have also shown that when monkeys are fed mono-unsaturated and poly-unsaturated fat diets, they have similar concentrations of LDL cholesterol, and these values are significantly lower than the LDL values from animals that are fed saturated fats (Rudel et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 15:2101-2110, 1995).
Therefore, a vegetable oil low in total saturates and high in mono-unsaturates and/or poly-unsaturates would provide significant health benefits to all consumers. The beneficial effects of oils high in poly-unsaturated fatty acids extend beyond lowering LDL cholesterol. For instance, linoleate and linolenate are essential fatty acids in human diets, rendering any edible oil high in these fats a useful nutritional supplement. Certain plants naturally possess high levels of poly-unsaturated fatty acids. This is exemplified by linseed oil, which is derived from the Flax plant (Linum usitatissimum) and contains over 50% linolenic acid. The oil content of flax is comparable to canola (around 40% dry weight of seed), however, high yields are only obtained in warm temperatures or subtropical climates. In addition, flax is highly susceptible to rust infection in the U.S. Therefore, even though natural plant sources of high poly-unsaturates exist, they are not always useful for large scale oil production. It would be commercially useful if a common crop such as canola, soybean or corn could be genetically transformed in such a way to minimize saturated fatty acid content.
To this effect, mutation-breeding programs have shown some promise in altering the levels of poly-unsaturated fatty acid levels in the edible oils of agronomic species. Examples of commercially grown varieties are high (85%) oleic sunflower and low (2%) linolenic flax (Knowles, (1980) pp. 35-38 in Applewhite, T. E., Ed., World Conference on Biotechnology for the Fats and Oils Industry Proceedings, American Oil Chemists' Society). However, these breeding programs are difficult to maintain and yields are often low. Hence, the option of production of transgenic plants is a desirable alternative for altering the content of saturated fats.
The enzymes of the fatty acid biosynthetic pathways are useful in creating transgenic plants that have altered fatty acid content. The β-ketoacyl-ACP (acyl carrier protein) family of synthase enzymes (also referred to herein as KAS) is especially attractive for plant transformation due to their indispensable role in fatty acid synthesis. To summarize their functions briefly, KAS III catalyzes the condensation of acetyl-CoA and malonyl-ACP to yield acetoacetyl-ACP in the first elongating reaction, KAS I utilizes saturated C2-C14 and unsaturated C16:1-C18:1 acyl-ACPs as substrates for condensation with a C2 unit derived from malonyl-ACP; KAS II carries out the final extension step of unsaturated fatty acid biosynthesis (C16:1 to C18:1) by utilizing C14:0 and C12:1-C16:1 acyl-ACPs. KAS IV has a substrate specificity between those of KAS III and KAS I, and is a medium chain specific condensing enzyme. See Siggaard-Andersen et al., Proc. Natl. Acad. Sci., Vol. 91, pp.11027-11031, November 1994, and Dehesh et al., Plant J, 15(3):383-390, August 1998.
To elaborate, the biosynthesis of fatty acids is a complex process, involving numerous enzymes and multiple plant compartments. The production of fatty acids in plants begins in the plastid with the reaction between acetyl-CoA and malonyl-ACP to produce butyryl-ACP. Elongation of acetyl-ACP to 16-and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP, reduction of the keto-function to an alcohol, dehydration to form an enoyl-ACP, and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP. KAS I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas KAS II catalyzes the final elongation to stearoyl-ACP (C18:0). The longest chain fatty acids produced by the fatty acid synthases are typically 18 carbons long. A further fatty acid biochemical step occurring in the plastid is the desaturation of stearoyl-ACP (C18:0) to form oleoyl-ACP (C18:1) in a reaction catalyzed by a delta-9 desaturase.
Once the C18:1-ACP has been formed, the products undergo de-esterification, which allows movement into the cytoplasm, wherein they are incorporated into the “eukaryotic” lipid biosynthesis pathway. This occurs in the endoplasmic reticulum, which is responsible for the formation of phospholipids, triglycerides and other neutral lipids. Following transport of fatty acyl CoA's to the endoplasmic reticulum, subsequent sequential steps for triglyceride production can take place. For example, polyunsaturated fatty acyl groups such as linoleoyl and a-linolenoyl, are produced as the result of sequential desaturation of oleoyl acyl groups by the action of membrane-bound enzymes. Triglycerides are formed by action of the 1-, 2-, and 3-acyl-ACP transferase enzymes glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase. Alternatively, fatty acids are linked to glycerol-3-phosphate (prokaryotic path), further unsaturated, and used for synthesis of chloroplast lipids. A portion of cytoplasmic lipids returns to the chloroplast. The preferential use of either eukaryotic or prokaryotic pathway depends on the particular plant species. The fatty acid composition of a plant cell is a reflection of the free fatty acid pool and the fatty acids (fatty acyl groups) incorporated into triglycerides as a result of the acyltransferase activities.
The properties of a given triglyceride will depend upon the various combinations of fatty acyl groups in the different positions in the triglyceride molecule. In general, vegetable oils tend to be mixtures of different triglycerides. The triglyceride oil properties are therefore a result of the combination of triglycerides which make up the oil, which are in turn influenced by their respective fatty acyl compositions.
There have been attempts to generate transgenic plants that would exhibit altered fatty acid levels for purposes of providing better nutritional value and herbicide and/or cold resistance. For instance, transformation of plants with maize acetyl CoA carboxylase or delta-15 desaturase can alter the fatty acid content in the plants. See U.S. Pat. Nos. 6,222,099 and 5,952,544 respectively. Transformation of plants with maize acetyl CoA carboxylase gene allows for altering the total oil content in the plant. However, this process does not provide a method for specifically increasing only the levels of unsaturated fats, which would in turn decrease the levels of saturated fats in the transgenic plants. U.S. Pat. No. 5,500,361 describes a nucleotide sequence that encodes for a β-ketoacyl-ACP synthase II enzyme. However, the disclosed compositions are limited in their use. Similarly, U.S. Pat. No. 6,200,788, also discloses a nucleotide sequence that encodes for a specific β-ketoacyl-ACP synthase II enzyme and has the same limitations.
Depending upon the intended oil use, various different oil compositions are desired. For example, edible oil sources containing the minimum possible amounts of saturated fatty acids are desired for dietary reasons and alternatives to current sources of highly saturated oil products, such as tropical oils, are also needed. Furthermore, oils compositions containing rare or exotic fatty acid species having nutritidnal benefits are also needed in the art. To this end, therefore, novel vegetable oils compositions and/or improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources, are needed.
Accordingly, a need still exists to produce transgenic plants that would possess lower levels of saturated fats compared to the levels in untransformed plants. These transgenic plants would provide a valuable nutritional supplement, especially if the oils extracted from them were incorporated into diets of people who were at risk for or suffered from cardiovascular diseases. Thus a need exists for a multi-functional KAS synthase that can effectively alter the saturated fatty acid content of a commercial plant. Thus, the identification of nucleic acid sequences encoding enzymes capable of producing altered saturated fatty acid compositions in host cells is needed in the art. Ultimately, useful nucleic acid constructs having the necessary elements to provide a phenotypic modification and host cells containing such constructs are needed.